In combination with sequencing of genomes, automated DNA synthesizers have given researchers the possibility to acquire almost any genetic element as an oligonucleotide and, at least for short oligonucleotides, at a fairly low cost. Two current limitations relate to: I) The production of large amounts of high quality oligonucleotides and II) The synthesis of very long oligonucleotides (200-1000 nucleotides). Concerning the former issue, the quality of the oligonucleotides can be affected by factors related to the chemical synthesis method, including depurination and dG to dA transitions. The maximum length of the synthesized oligonucleotides is currently around 150 nucleotides, with the yield and quality dropping as the length increases.
The main current uses of oligonucleotides are as primers for the polymerase chain reaction (PCR), for reverse transcription PCR (rtPCR), for sequencing, and as substrates for different enzymatic reactions. In recent years, new techniques have appeared which demand high quality, 5′ phosphorylated oligonucleotides of 70-100 nucleotides in length (e.g. padlock probes). Such probes may well cost 100-200$, taking the extra cost for 5′ phosphorylations (25-35$) and HPLC purifications (25-35$) into account.
These oligonucleotides can to some extent be amplified enzymatically by known methods, e.g. cloning, PCR, rolling circle amplification or Circle-to-circle amplification (C2CA). However, all of these techniques have limitations.
Cloning
Cloning is a time consuming technique where a double stranded DNA sequence is inserted into a plasmid, transformed into an appropriate host organism e.g. bacteria or yeast, the organism grown, and the DNA purified. This is then followed by isolation of the DNA fragment of interest by restriction endonucleases. The technique is laborious, is more suitable for the production of double stranded DNA and at the same time a lot of non-useful DNA is co-amplified and, in particular for the production of shorter DNA segments, the production of non-useful (vector)-DNA will be dominant. Furthermore a laboratory classified for gene modified organisms is required.
PCR
PCR is based on the amplification of a double stranded DNA fragment by the use of a thermostable DNA polymerase and primers complementary to the DNA fragment. Amplification of the DNA fragment takes place by alternating heating and cooling. This technique is more suitable for the production of double stranded DNA and requires a primer for each amplified DNA strand. Furthermore, since the product created is used as template for the next rounds of amplification, mis-incorporation of a nucleotide early in the process will be amplified exponentially along with the desired product.
Rolling Circle Replication
In vitro rolling circle replication traditionally uses a circular single stranded DNA oligonucleotide as a rolling-circle-template and a short oligonucleotide as a primer. The addition of a DNA polymerase and dNTPs starts the polymerization. As the rolling-circle-template is endless, the product will be a long single stranded DNA molecule composed of tandem repeats complementary to the rolling-circle-template. In contrast to the PCR reaction, a falsely incorporated nucleotide will not be further amplified as the circular oligonucleotide is template for each round of amplification.
US 2003/0087241 describes the method of “Rolling circle amplification” in which a rolling circle product can be designed in a way, that allows it to fold into distinct hairpin structures containing a binding site for a restriction endonuclease. The rolling circle product can therefore, by a restriction endonuclease, be turned into monomers. Limitations to this technique include that it does not amplify the circular oligonucleotide, but rather replicate the complementary sequence, and that it only provides one round of amplification.
Circle-to-Circle Amplification
Circle-to-circle amplification (C2CA) is a method for the amplification of a single stranded DNA sequence, based on successive rounds of rolling circle DNA synthesis. The DNA sequence is circularized using an external template for ligation. Following rolling circle DNA synthesis, a long tandem repeat complementary to the start DNA sequence is synthesized. By hybridization of an oligonucleotide to the rolling circle product, at a position containing a restriction site, the single stranded tandem repeat can be turned into monomers by addition of a restriction endonuclease. Following a second circularization, rolling circle DNA synthesis, hybridization of a new oligonucleotide (complementary to the first one) and cutting with a restriction endonuclease, an amplification of the start DNA sequence has taken place through two rounds of rolling circle DNA synthesis (Dahl F et al., Proc Natl Acad Sci USA. 101(13), 4548-53 (2004)).
This technique requires the additional production of oligonucleotides for cleavage of the rolling circle product, and, even more importantly, it does not provide free design of the ends of the DNA sequence to be amplified as they are defined by the presence of a binding/cleavage site for a restriction endonuclease.
Taken together, there is a need for improved techniques for the production of large quantities of high-quality oligonucleotides to eliminate the limitations associated with the existing techniques as outlined above. The invention disclosed here represents just that, providing large quantities of phosphorylated high quality oligonucleotides, e.g. exhibiting superior performance for circle formation.